Certain alterations of proteins involved in mitogenic signaling are known to exert profound effects on cellular behavior, including malignant transformation. Our overall objective is to explore the molecular bases of cancer, approaching this problem through the study of normal and aberrant functioning of molecules that participate in the transduction of proliferative signals. [unreadable] [unreadable] 40% Effort. Molecular dissection of the pathway linking growth factor receptors to the nucleus: their role in normal cell growth and cancer.[unreadable] [unreadable] RGS-containing RhoGEFs: the link between transforming G proteins and RhoA: Prior work by our laboratory and others indicates that G protein-coupled receptors (GPCRs) coupled to the G13 family of G proteins stimulate the small GTPase RhoA through the direct interaction of Galpha12/13 with a group of Rho guanine nucleotide exchange factors (GEFs) comprised of PDZ-RhoGEF (PRG), LARG, and p115-RhoGEF (p115). These GEFs are characterized by the presence of a regulator-of-G-protein-signaling (RGS) domain by which they bind receptor-stimulated Galpha12/13, thus providing a molecular bridge for the activation of RhoA by Galpha12/13. We have engineered animals in which the gene for PRG or LARG has been disrupted. While no obvious phenotype was initially observed in animals deficient in either gene, we have recently observed that mice deficient in LARG are resistant to salt-induced hypertension, supporting a key role for G12/13 coupled receptors and RhoA in blood pressure control. Furthermore, mice lacking both PRG and LARG die at about embryonic day E9.5. Their major phenotype is that they have deficient embryonic blood vessels, supporting the relevance of the Galpha13-PRG/LARG axis in developmental angiogenesis and endothelial cell function.[unreadable] [unreadable] Multiple LRP6 phospho-acceptor sites are required for Wnt signaling: Wnt is a secreted glycoprotein that functions as a paracrine mediator of developmental patterning and cell proliferation, and whose abnormal activity contributes to many human cancers. We have recently shown that GPCRs involved in tumor progression converge with the Wnt-initiated pathway to promote the activation of the transcription factor b-catenin, thereby promoting cell proliferation. Wnt binds to a GPCRlike receptor, Frizzled, and LRP5/6, which are members of the low-density-lipoprotein-like receptor protein family, and this causes b-catenin accumulation by a poorly understood mechanism. We hypothesized that five phosphorylatable motifs of LRP6 (PPPS/TP), that are conserved from flies to humans, may contribute to stimulate b-catenin. We engineered full-length LRP6 mutants harboring multiple combinatorial mutations in its five PPPS/TP sites. This revealed that the five PPPS/TP sites are not functionally equivalent, and that they each contribute independently to LRP6 function in the canonical Wnt-b-catenin pathway.[unreadable] [unreadable] 40% Effort. Molecular basis of developmental and tumor-induced angiogenesis. [unreadable] [unreadable] Molecular mechanisms by which Semaphorins and Plexins promote angiogenesis: We have shown that Sema4D elicits a pro-angiogenic phenotype in endothelial cells by promoting the activation of the PI3K-Akt signaling pathway through its receptor, Plexin-B1. By the use of a receptor chimeric approach, Plexin-B1 mutants, knock down strategies, and dominant negative inhibitors, we found that this response is dependent upon the activation of RhoA and its downstream target, Rho kinase (ROK). Furthermore, we have recently observed that Plexin-B1 utilizes RhoA and ROK to activate an integrin-dependent signaling network that stimulates Pyk2, PI3K, Akt, and ERK, and endothelial motility.[unreadable] [unreadable] The molecular basis of VEGF-induced endothelial cell permeability, and the counteracting effect of angiopoietin-1: VEGF is a pleiotropic factor for endothelial cells that plays a central role in both vasculogenesis and angiogenesis. Deregulated VEGF expression also contributes to the aberrant growth of most solid tumors by promoting their neo-vascularization. In an effort aimed at exploring the mechanism by which VEGF stimulates the proliferation, migration, and survival of endothelial cells, we began investigating how VEGF induces vessel leakiness, its first described function. Last year, we identified a novel signaling mechanism, involving the sequential activation of Src, Vav2, Rac1, and Pak1, by which VEGF promotes the rapid removal of a key cell-cell adhesion molecule, VE-cadherin, from the cell surface, leading to the disassembly of endothelial cell junctions and enhanced permeability. These findings opened new therapeutic opportunities for the treatment of many human diseases that involve pathological vessel leakiness. In this regard, Angiopoietin 1 (Ang1) represents the only known natural blocker of VEGF-induced blood vessel leakiness. How Ang1 exerts its anti-vascular permeability effects remained unknown. Surprisingly, we found that Ang1 potently inhibits the activation of Src, the most upstream component of the biochemical route linking VEGF receptor to VE-cadherin phosphorylation and internalization. The further dissection of the intervening mechanism revealed that Ang1 stimulates the activation of the small GTPase RhoA and its direct downstream target, Diaphanous, which in turn binds Src. Ultimately, our findings indicate that Ang1 causes the sequestration of Src through Diaphanous, thereby depriving VEGF receptors from an essential molecule required to initiate a signaling route that promotes the disruption of endothelial cell-cell contacts. These studies suggest that inhibitors of the mDia-Src signaling axis may represent potential anti-vascular permeability therapeutic agents.[unreadable] [unreadable] 20% Effort. AIDS-associated Kaposi s sarcoma: molecular mechanisms.[unreadable] [unreadable] A NF-kappaB gene expression signature contributes to KSHV vGPCR-induced direct and paracrine neoplasia: Among the AIDS-associated malignancies, Kaposis sarcoma (KS) is the most common cancer arising in HIV-infected patients. The Human herpesvirus 8 (HHV-8) or KS associated herpesvirus (KSHV) is the infectious cause of KS. In prior studies, we have developed a high throughput in vivo endothelial specific retroviral gene transfer system, and used it to express candidate KSHV oncogenes in mice. Surprisingly, among the many KSHV genes tested, only one gene, a constitutively active GPCR, vGPCR, was able to promote the development of visible vascular tumors that strikingly resembled human KS lesions. Using a full-genome microarray analysis we found that vGPCR promotes a dramatic change in gene expression in endothelial cells either by acting directly in vGPCR-expressing cells, or indirectly through the release of soluble factors. By using gene set enrichment analysis of the microarray data, we found that NF-kappaB signaling is potently triggered by vGPCR expression and in cells exposed to vGPCR-induced secretions, thus mimicking its paracrine effect. Indeed, we observed that vGPCR activates the NF-kappaB pathway effectively, and NF-kappaB activation is a prominent feature in both human and experimental KS. Of interest, constitutive NF-kappaB signaling is not sufficient to promote endothelial cell transformation. However, using genetic approaches to block NF-kappaB we found that this transcription factor is strictly required for vGPCR-induced direct and paracrine transformation. Taken together, these results strongly support the role of NF-kappaB and its regulated genes in KS pathogenesis.